At the creation and application of biosensors appeared a number of problems which are: 1) optimization of process connected with stabilization of the structure of biomolecules at the integration with the transducers to preserve their maximum activity and 2) search of approaches to accomplish repeated analysis of substances which are irreversible inhibitors of activity of above mentioned molecules. In this article the results obtained in time of solving of these problems at the usage of enzymes as sensitive bilayer of biosensors are analyzed. For stabilization of the structure of such enzymes as:(beta) - glucose oxydase, urease, cholinesterases during their immobilization the following approaches were examined: 1) usage of one or combination of chemical substances: protein, saccharose, ethylendiamine tetraacetic acid (EDTA), glycerol, ditiotrie-tole (DTT) and specific substrates or their homologues; 2) variation of covalent crosslinking methods including usage of bifunctional reagents in aqueous and vaporous phases; 3) change of time of the influence of this reagent. Optimization of these parameters can allow to preserve about 70-80 percent of initial enzyme activity at the usage of such bifunctional reagent as glutaraldehyde. For repeated analysis of phosphoroganic pesticides and heavy metal ions which are irreversible inhibitors of enzymes the following approaches were applied: 1) treatment of enzyme membrane by special reactivating substances ; 2) usage of easily replaceable enzymatic membrane. It was shown that the last way is more preferable, particularly if alginate gel or nitrocellulose is used for direct enzyme immobilization or preparation of separated biomembrane respectively. Standard deviation of sensor responses for different membrane castings did not exceed 10 percent. At the same time this parameter changed more strongly after even one use of reactivating reagents.